Because at least a portion of what is described below was sponsored under a federal contract, the federal government may retain certain rights in the invention.
The invention relates to an antibiotic purification method.
Antibiotic sales total billions of dollars per year, and have increased. Antibiotics are prepared from biological sources by extraction; by chemical synthesis; or by a combination of these methods. Because of the large market for antibiotics, there is a long-felt need for improved methods of recovering antibiotics from biological sources. An improved method may possess any one or more of the following features (presented by way of example and not of limitation): lower cost; greater yield; greater purity of the final product; use of starting materials or reagents that are more readily available; production of less harmful waste-stream or by-products; etc.
While antibiotics are thought of as means to control infectious disease, antibiotics are also useful in the treatment of chronic disease such as cancer. In particular, certain classes of antibiotics which are cytotoxic to certain cancer cells, and hence are useful as antineoplastic chemotherapeutic agents, are either in use or under active investigation as anticancer pharmaceuticals. See generally Remers, William A., The chemistry of antitumor antibiotics, Vols. 1 and 2 (1979, 1989). There are several quinone-containing agents which cause DNA damage and which have therefore been useful as antineoplastic agents. See Begleiter, A., Clinical applications of quinone-containing alkylating agents. Frontiers in Bioscience 5, e153-171 (2000). However, there has been interest in other agents which might act against other cellular targets, such as heat shock protein 90 (Hsp90). Hsp90 is a chaperone protein that has been found to be associated with cell proliferation proteins, such as certain protein kinases. Hsp90 is found at higher levels in tumor cells than in normal cells. Members of the benzoquinoid ansamycin class of drugs, such as geldanamycin, herbimycin A, and macbecin II, have been shown to bind specifically to Hsp90 and affect Hsp90 function, and are regarded as first generation Hsp90 antagonists. Among the benzoquinone ansamycins, geldanamycin in particular has been recognized and studied as an Hsp90 inhibitor. Because Hsp90 and similar proteins are regarded as targets for antineoplastic chemotherapy, geldanamycin""s effectiveness at inhibiting tumor growth has been studied, with results showing 13 nM geldanamycin achieving growth inhibition of 50% in highly responsive cell lines. There has therefore been interest in preparation and purification of geldanamycin for laboratory or clinical study. See generally Neckers, L., et al. Geldanamycin as a potential anti-cancer agent: Its molecular target and biochemical activity. Investigational New Drugs 17: 361-373 (1999).
Antibiotics tend to have complex structures, making their total synthesis somewhat difficult. When a large market has not yet been established for an antibiotic, it is not necessarily cost-effective to produce an antibiotic routinely by total synthesis. Instead, the antibiotic may be prepared by extraction from a biological source or, in some cases, semi-synthetically.
Therefore it is known in the art that certain antibiotics can be recovered from fermentation broths. See, for example U.S. Pat. Nos. 5,188,945; 5,591,438; 5,942,611.
In the art, the benzoquinoid ansamycin antibiotic geldanamycin has been prepared from the culture filtrate of Streptomyces hygroscopicus var. geldanus var. nova, through the use of an initial butanol extraction followed by subsequent steps employing the halogenated organic solvent chloroform. Geldanamycin has been shown to be soluble in alcohols and aliphatic chlorinated solvents such as chloroform; to be less soluble in acetone, benzene, and ethyl acetate; to be only slightly soluble in water; and to decompose easily with acid, base, or heat in the presence of oxygen. See DeBoer, C., et al. Geldanamycin, a new antibiotic. J. Antibiot. 23, 442 (1970). Geldanamycin also undergoes photolytic degradation in both aqueous and nonaqueous solutions. See Supko, J. G., et al., Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother Pharmacol 36: 305-315 (1995).
There may be certain limitations to extractions that rely on halogentated organic solvents. Certain halogenated organic solvents such as chloroform may be regarded as hazardous materials and hence may give rise to a waste-stream containing hazardous materials and may need to be thoroughly removed from an end-product that might be administered to humans.
Commonly used liquid-solid extraction processes may employ the Soxhlet apparatus. Such processes may take a long time and produce high-volume, dilute solutions which typically must be concentrated.
On the basis of the art it can be seen that it might reasonably be desired to devise a method for recovering a benzoquinoid ansamycin antibiotic from a fermentation broth, which method does not rely on halogenated organic solvents and which method does not needlessly subject the desired product, geldanamycin, to degradation due to acid, base, or heat.
The present invention provides inter alia a process for purifying a benzoquinoid ansamycin antibiotic, said process comprising contacting a mixture comprising the antibiotic with a fluid comprising supercritical carbon dioxide.
The present invention also provides inter alia a process for purifying a benzoquinoid ansamycin antibiotic from a fermentation broth that contains the antibiotic, said process comprising (a) separating the fermentation broth into a particulate phase and a liquid phase, and (b) extracting the antibiotic from the particulate phase.
The present invention also provides inter alia geldanamycin purified by a process comprising contacting a mixture comprising geldanamycin with a fluid comprising supercritical carbon dioxide.
As used in connection with the present invention, xe2x80x9cpurifyingxe2x80x9d means substantially separating a desired composition from a mixture that contains not only the desired composition but also at least one other composition. For example, evaporation is a process for purifying sodium chloride from an aqueous solution of sodium chloride. In this example, after evaporation, that is, after the process for purifying sodium chloride has been performed, the desired sodium chloride is obtained substantially free of water. However, a relatively small quantity of water may yet remain with the desired sodium chloride even after the process has been performed. Therefore, purifying results in a composition""s being separated from at least one other composition in a mixture but need not result in a composition""s being separated from every molecule of every other composition in the mixture. For example, crystallization is a process for purifying a desired composition that can form a crystalline solid from a mixture that contains not only the desired composition but also a crystallization solvent and an impurity. The crystalline solid obtained by crystallization may contain relatively small amounts of the crystallization solvent and may also contain relatively small amounts of the impurity.
As used in connection with the present invention, xe2x80x9cextractingxe2x80x9d means contacting a matrix with a fluid, wherein said contacting transfers a desired composition from the matrix to the fluid.
In seminal work concerning the benzoquinoid ansamycin antibiotic geldanamycin, DeBoer, C., et al. Geldanamycin, a new antibiotic. J. Antibiot. 23, 442 (1970), the filtrate of S. hygroscopicus culture medium was extracted with butanol to recover impure geldanamycin. Subsequent steps in the DeBoer work included crystallization, dissolution of the crystals in chloroform, filtration, and another crystallization. In later work performed by other investigators at the National Cancer Institute (NCI) Frederick Cancer Research and Development Center, lot #100904 was prepared by initial steps including centrifugation of the whole broth and extraction of the supernatant with ethyl acetate, the solids resulting from the centrifugation of the whole broth being discarded.
In part on the basis of the results of preparation of lot #100904, it was recognized in the art that existing methods of recovering geldanamycin from fermentation broth were inadequate. Moreover, on the basis of the DeBoer work and that of NCI, the art taught away from recovering geldanamycin from the solids resulting from the centrifugation of the whole broth.